Methane- and methanol-oxidizing bacteria (methylotrophs) are capable of growth on methane or methanol as their sole source of carbon and energy. They are ubiquitous in the natural environment and plays a significant role in carbon cycling in specific habitats. Since they are net consumers of volatile carbon, they have a direct impact on the quality of water systems. In addition, they are also capable of cooxidizing a variety of other compounds including straight-chain and cyclic hydrocarbons, as well as halogenated hydrocarbons. Since many of these compounds are toxic, methylotrophs appear to play an important role in detoxification in nature, and may have potential for the development of detoxification processes for contaminated aquifers and industrial waste streams. In order to understand the role that methylotrophs play in maintaining a healthy environment it is important to study this group in detail. This laboratory is particularly interested in gene organization and expression in methylotrophs, and the long term goal of this project is to understand the regulation of C-1 functions in these organisms. Under previous NIH support mutagenesis and gene transfer systems have been developed for methylotrophs by taking advantage of recombinant DNA techniques, and these have been used to study C-1 metabolism in both facultative methanol-utilizers and obligate methane-utilizers. It is now proposed to continue this effort, using a combination of genetic and physiological approaches. The methanol oxidation (Mox) system will be the focus of this work, and it is proposed to define the remaining Mox functions of the facultative methanol-utilizer. Methylobacterium AM1 both genetically and physiologically. Promoter-lacZ fusions for key Mox genes will then be used to study regulation of this system in both batch and continuous culture. In addition, similar fusion will be constructed for assimilation genes, and their regulation will be studied in batch culture for comparison to the Mox system. Using the information, mutants an gene probes generated in the Methylobacterium AM1 studies, Mox functions and their regulation will also be defined in the obligate methane-utilizer, Methylomonas albus BG8.